PROJECT SUMMARY/ABSTRACT Cryptococcal meningitis (CM) is a severe central nervous system (CNS) infection caused by the fungal pathogen Cryptococcus neoformans, primarily in people with compromised immune systems. CM kills 181,000 people annually, with the largest burden on AIDS patients. It is the second most common killer of AIDS patients in sub-Saharan Africa, behind only tuberculosis. Both the treatment and diagnosis of CM is complicated by a well-documented variability in disease presentation. Changes in patient outcome have been associated, in part, with the genotype of C. neoformans. We performed a preliminary genome wide association study (GWAS) on 40 whole genomes of clinical isolates with associated patient data. The GWAS revealed an association between single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) in C. neoformans genes and patient outcome. We deleted a sub-set of these genes and showed they play a previously uncharacterized role in virulence. Furthermore, there is a documented association between structural variation in the C. neoformans genome and patient outcome. This proposal will test the hypothesis that individual nucleotide variations cause changes in patient disease presentation. Aim 1 will identify SNPs/INDELs in a large population of C. neoformans clinical isolates by using amplicon sequencing and will use long-read sequencing to characterize structural variants in the clinical isolates. We will compare these data to patient outcome to identify clinically relevant genomic variants. Aim 2 will determine the role of the nucleotide variations in a biological context by exchanging gene alleles in virulent or avirulent genetic backgrounds and then analyzing changes in pathogenesis. Aim 2 will define the biological function of the observed genomic differences through characterization of novel genes identified in the preliminary GWAS analysis. Finally, we will introduce single nucleotide variations into the reference gene alleles using CRISPR to define the impact of single genetic changes. The proposed research is innovative in that it is the first to compare single nucleotide polymorphisms in C. neoformans and differences in patient outcome. The proposed research will 1) determine the role of SNP/INDELs on C. neoformans phenotype; 2) identify unknown virulence factors; and 3) build a diagnostic method for identifying the infecting C. neoformans strain, leading to targeted therapy and improved patient survival.